Journal: Thoracic Cancer

Article Title: Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR ‐6838‐5p

doi: 10.1111/1759-7714.14820

Figure Lengend Snippet: Knockdown of circ_0001667 inhibited proliferation and angiogenesis in breast cancer cells. MDA‐MB‐231 and BT549 cells were transfected with si‐NC or si‐circ_0001667. (a) qRT‐PCR assay for silencing efficiency of si‐circ_0001667. (b‐d) CCK8 and EdU assays to detect proliferation of breast cancer cells. Scale bar: 50 μm. (e and f) Flow cytometry was used to determine the cycle distribution of breast cancer cells. (g) Colony formation assay was used to assess the colony formation ability. (h) Western blot to measure the protein levels of PCNA and Ki67. (i) Tube formation assay was used to analyze angiogenesis of breast cancer cells. (j) Western blot showing the expression levels of VEGFA and FGF2 in breast cancer cells. * p < 0.05.

Article Snippet: The separated proteins were electrotransferred to PVDF membrane (Beyotime), closed with 10% skimmed milk, and incubated with primary antibodies (Abcam) against PCNA (ab18197, 1:1000), Ki67 (ab254123, 1:5000), VEGFA (ab46154, 1:2000), FGF2 (ab208687, 1:1000), CXCL10 (ab9807, 1:2000), or β‐actin (ab6276, 1:5000).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Flow Cytometry, Colony Assay, Western Blot, Tube Formation Assay, Expressing

Journal: Thoracic Cancer

Article Title: Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR ‐6838‐5p

doi: 10.1111/1759-7714.14820

Figure Lengend Snippet: Downregulation of miR‐6838‐5p attenuated the effect of circ_0001667 knockdown on cell proliferation and angiogenesis. (a) qRT‐PCR analysis of miR‐6838‐5p levels in cells transfect with anti‐miR‐6838‐5p or anti‐miR‐NC. (b‐j) MDA‐MB‐231 and BT549 cells were transfected with si‐NC, si‐circ_0001667, si‐circ_0001667 + anti‐miR‐NC or si‐circ_0001667 + anti‐miR‐6838‐5p. (b‐d) CCK‐8 and EdU assays detected cell proliferation. (e and f) Flow cytometry determined cell cycle distribution. (g) The colony formation assay assessed the colony formation ability. (h) Western blot measured the protein levels of PCNA and Ki67. (i) Tube formation test analyzed angiogenesis. (j) Western blot showed the expression levels of VEGFA and FGF2. * p < 0.05.

Article Snippet: The separated proteins were electrotransferred to PVDF membrane (Beyotime), closed with 10% skimmed milk, and incubated with primary antibodies (Abcam) against PCNA (ab18197, 1:1000), Ki67 (ab254123, 1:5000), VEGFA (ab46154, 1:2000), FGF2 (ab208687, 1:1000), CXCL10 (ab9807, 1:2000), or β‐actin (ab6276, 1:5000).

Techniques: Knockdown, Quantitative RT-PCR, Transfection, CCK-8 Assay, Flow Cytometry, Colony Assay, Western Blot, Expressing

Journal: Thoracic Cancer

Article Title: Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR ‐6838‐5p

doi: 10.1111/1759-7714.14820

Figure Lengend Snippet: Overexpression of CXCL10 reversed the effects of miR‐6838‐5p overexpression on cell proliferation and angiogenesis. (a and b) Validation of CXCL10 overexpression by qRT‐PCR and western blot. (c–k) MDA‐MB‐231 and BT549 cells were transfected with miR‐NC, miR‐6838‐5p, miR‐6838‐5p + pcDNA or miR‐6838‐5p + CXCL10. (c–h) CCK8 assay, EdU assay, flow cytometry and colony formation assay detected cell proliferation. (i) Western blot analysis for PCNA and Ki67 levels. (j) Tube formation test detected angiogenesis. (k) Western blotting quantified the levels of VEGFA and FGF2. * p < 0.05.

Article Snippet: The separated proteins were electrotransferred to PVDF membrane (Beyotime), closed with 10% skimmed milk, and incubated with primary antibodies (Abcam) against PCNA (ab18197, 1:1000), Ki67 (ab254123, 1:5000), VEGFA (ab46154, 1:2000), FGF2 (ab208687, 1:1000), CXCL10 (ab9807, 1:2000), or β‐actin (ab6276, 1:5000).

Techniques: Over Expression, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, EdU Assay, Flow Cytometry, Colony Assay

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: KLC1 interaction with DENV NS1 in infected C6/36 cells. ( a ) Colocalization assays of KLC1 and DENV NS1 (Pearson=0.52). ( b ) PLA of KLC1 and DENV NS1. Mock-infected cells treated with anti-KLC1 ( mock ) and infected cells with primary anti-KLC1 antibody omitted ( control ) were included as negative controls. Crop in (a) and (b); enlargement of the cells enclosed in the yellow square. ( c ) Immunoprecipitation assays. Cell lysates were immunoprecipitated with anti-NS1 antibodies bound to Sepharose beads. The presence of DENV KLC1 in the immunoprecipitant was revealed by western blot. Changes in protein migration observed in the immunoprecipitate are due to elution buffer composition and were consistently observed in experimental replicas. In all cases, mock and infected cells were harvested at 24 hpi. C6/36 average cell diameter=12.5 µm. Bar=5 µm. Representative images of at least three independent experiments are shown.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Infection, Control, Immunoprecipitation, Western Blot, Migration

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: In silico modelling of mosquito KLC1. ( a ) AlphaFold 3.0 modelling of the KLC1 of A. albopictus . TRP domain shown in blue. (b) Superimposition of the TPR cargo binding domain of mosquito (blue) and human (red) KLC1. ( c ) Modelled interaction of the TPR (blue) and NS1 dimers (orange). pTM: predicted template modelling. ipTM: interface predicted template modelling. RMSD: root mean square deviation. Circular arrow=180° image rotation.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: In Silico, Binding Assay

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Immunoelectron microscopy of C6/36 cells infected with DENV. KLC1 was labelled with 30 nm (red arrowheads), and NS1 with 15 nm (green arrowheads) gold particles. (a) Mock-infected cells; (b) and (c) infected cells. Cells were fixed at 24 hpi. KLC1 and NS1 were found to colocalize around vacuole-like structures. N=cell nuclei. Blue arrowheads=lipid cores.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Immuno-Electron Microscopy, Infection

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: KLC1 expression levels in DENV-infected cells. Cell lysates of mock- and DENV-infected cells were analysed by western blot. Relative KLC1 protein levels are expressed as a ratio to β -actin used as load control. Representative images of three independent experiments are shown. * P ≤0.005.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Expressing, Infection, Western Blot, Control

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect on DENV replicative cycle of silencing the expression of KLC1. ( a ) Transfection of C6/36 cells with siRNA specific for KLC1 or an NR siRNA. Cells were lysed 24 hpt, and the level of protein expression was analysed by western blot. Relative KLC1 protein levels are expressed as a ratio to GAPDH. ( b ) Expression levels of NS1 in DENV-infected cells silenced or not for KLC1 and lysed 24 hpi. NS1 levels are expressed as a ratio to GAPDH. ( c ) Amount of secreted NS1 in cells silenced or not for the expression of KLC1. Secreted NS1 was measured by an in-house ELISA, expressed as a percentage of the control condition, taken as 100%. ( d ) Virus genome levels. RNA levels were determined by qRT-PCR. ( e ) Virus yield. Virus progeny was determined by plaque assay. Cells were lysed and supernatants collected at 24 hpi. * P ≤0.005. **** P ≤0.001. n ≥3.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Expressing, Transfection, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Control, Virus, Quantitative RT-PCR, Plaque Assay

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect on DENV RC of cells silenced for KLC1. Cells transfected with an NR siRNA. Cells transfected with a KLC1-specific siRNA. Cells were fixed at 24 hpi. Bar=5 µm. Representative images of three independent experiments are shown.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Transfection

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect of the KINTAG peptide on the replication of DENV. ( a ) Fluorescence intensity of NS1 in cells transfected with KINTAG peptide fixed at 24 hpi. ( b ) Expression levels of NS1 in DENV-infected cells with KINTAG peptide 24 hpi. NS1 levels are expressed as a ratio to tubulin. ( c ) Amount of secreted NS1 in cells with KINTAG peptide. Secreted NS1 was measured by an in-house ELISA, expressed as a percentage of the control condition, taken as 100%. (d) Virus genome levels. RNA levels were determined by qRT-PCR. ( e ) Virus yield. Virus progeny was determined by plaque assay. Cells were lysed and supernatants collected at 24 hpi. ( f ) Comparison between 6SWU crystal structure (purple) and C6/36’s KLC1 TPR and KINTAG peptide interaction predicted by AlphaFold (blue); KINTAG peptide is shown in green. Bar=10 µm. * P ≤0.005. **** P ≤0.001. n ≥3.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Fluorescence, Transfection, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Control, Virus, Quantitative RT-PCR, Plaque Assay, Comparison

Journal: The Journal of General Virology

Article Title: Kinesin light chain 1 interacts with NS1 and is a susceptibility factor for dengue virus infection in mosquito cells

doi: 10.1099/jgv.0.002132

Figure Lengend Snippet: Effect of KLC1 silencing or function disruption on LD homeostasis. ( a ) Mock-infected cells silenced (siRNA) or not (NR) for KLC1 expression. ( b ) DENV-infected cells silenced or not for KLC1 expression. Cells were fixed at 24 hpi. ( c ) LD count in mock and infected cells, silenced or not for KLC1. The graph represents the LD count and area measured in pixels of 130 cells for each experimental condition. ( d ) Mock-infected cells transfected with KINTAG, fixed at 24 hpt. ( e ) LD count in KINTAG transfected cells. LD were stained with oil red-O. Bar=10 µm. Representative images of three independent experiments are shown. * P ≤0.005.

Article Snippet: Finally, the wells were washed four times with PBS 0.05% Tween-20, and 100 μl of ABTS substrate (Sigma, A-1888) with H2O2 0.3% was incubated for 1 h. A commercial recombinant DENV NS1 protein (R&D Systems), serially diluted, was used as a standard to build the calibration curve.

Techniques: Disruption, Infection, Expressing, Transfection, Staining

Journal: Sensors (Basel, Switzerland)

Article Title: Label-Free Electrochemical Biosensors for the Determination of Flaviviruses : Dengue, Zika, and Japanese Encephalitis

doi: 10.3390/s20164600

Figure Lengend Snippet: Survey of electrochemical label-free biosensors for Zika diagnostic.

Article Snippet: Microtiter plate precoated with ZIKV NS1/ Euroimmun anti-ZIKV IgM , Indirect ELISA , Antibody-HRP conjugates , DENV Ig M antibodies , 56% , West Nile virus , d, f , a, c , [ , ] .

Techniques: Diagnostic Assay, Virus, Polymer, Membrane, Indirect ELISA

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: (A) (B) Platelets were incubated for 1 h with DENV or ZIKV supernatant that contained different concentrations of NS1 as indicated (n = 4). (C) (D) Platelets were incubated with medium, 10 μg/ml NS1-containing DENV supernatant or NS1-depleted DENV supernatant (NS1Δ) for 1 h (n = 3). The percent fluorescence of P-selectin surface expression on platelets and annexin V binding to platelets were analyzed by FACSCalibur flow cytometry. *P<0.05, ***P<0.001; unpaired t-test (panels A and B), Kruskal-Wallis ANOVA (panels C and D).

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Incubation, Fluorescence, Expressing, Binding Assay, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: (A) Platelets were treated with BSA or DENV NS1 recombinant proteins (10 μg/ml) for the indicated time (n = 3). (B) Platelets were treated with different concentrations of DENV NS1 recombinant proteins for 1 h (n = 3). (C) Platelets were treated with BSA or DENV NS1 (10 μg/ml) (cotreated with or without 25 μg/ml anti-NS1 specific antibodies F(ab’) 2 fragment, 33D2 F(ab’) 2 , or the isotype-matched mouse antibodies F(ab’) 2 and cmIgG F(ab’) 2 ) for 1 h (n = 3). (D) Platelets were treated with BSA or DENV1-4 NS1 (10 μg/ml) for 1 h (n = 3). The percent fluorescence of P-selectin surface expression on platelets was analyzed by FACSCalibur flow cytometry. ***P<0.001; unpaired t-test (panels A and B), Kruskal-Wallis ANOVA (panels C and D).

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Recombinant, Fluorescence, Expressing, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: (A) Platelets were treated with BSA or DENV NS1 recombinant protein (10 μg/ml) for the indicated time (n = 3). (B) Platelets were treated with different concentrations of DENV NS1 recombinant protein for 1 h (n = 3). (C) Platelets were treated with BSA or DENV NS1 (10 μg/ml) (cotreated with or without 25 μg/ml 33D2 F(ab’) 2 , or cmIgG F(ab’) 2 ) for 1 h (n = 3). (D) Platelets were treated with BSA or DENV1-4 NS1 (10 μg/ml) for 1 h (n = 3). The percent fluorescence of annexin V binding to platelets was analyzed by FACSCalibur flow cytometry. *P<0.05, **P<0.01, ***P<0.001; unpaired t-test (panels A and B), Kruskal-Wallis ANOVA (panels C and D).

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Recombinant, Fluorescence, Binding Assay, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P<0.05, **P<0.01; Kruskal-Wallis ANOVA (panels B to panel F).

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Expressing, Control, Isolation, Western Blot, Binding Assay, Flow Cytometry, Bioprocessing, Knock-Out, Fluorescence

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: (A) Platelets were plated on poly-L-lysine-coated coverslips and treated with BSA or DENV NS1 (10 μg/ml) for 1 h. After fixation and permeabilization, platelets were stained with anti-CD61 polyclonal antibody and an anti-rabbit Alexa 594-conjugated antibody. (B) The light transmission of platelet-rich plasma (PRP) was measured in a Chrono-log aggregometer for 5 min after ADP (10 μM) or DENV NS1 (10 μg/ml) was added. (C) PRP was treated with BSA or DENV NS1 (10 μg/ml) (cotreated with or without 25 μg/ml 33D2 F(ab’)2, or cmIgG F(ab’)2) for 1 h and stimulated with ADP (2.5 μM). The light transmission of PRP was measured in a Chrono-log aggregometer, and (D) the maximum percentage of platelet aggregation was calculated by an AggRAM ™ platelet aggregation analyzer (n = 3). **P<0.01; Kruskal-Wallis ANOVA (panel D).

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Staining, Transmission Assay, Clinical Proteomics

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: (A) DENV-NS1-activated platelets were cultured with confluent HUVEC monolayers for 1 h, followed by washing and fixation. The adherent platelets were examined by confocal microscopy. CD61, a platelet marker, is stained in green, and HUVEC nuclei are stained with Hoechst (blue). (B) The adherent platelets on HUVEC monolayers in (A) were quantified in 100 HUVECs from three independent experiments and expressed as the mean fluorescence intensity (MFI) with ImageJ. (C) For the phagocytosis assay, DENV-NS1-activated platelets were cultured with PMA-activated THP-1 cells for 4 h. After fixation and permeabilization, the cells were stained with CD61 (green, a marker of platelets) and CD14 (red, a marker of monocytes/macrophages) and examined by confocal microscopy. The yellow dots represent the engulfed platelets, and the green dots represent the adherent platelets. (D) The average number of engulfed platelets per THP-1 cell was determined by counting 100 THP-1 cells per sample. The images were further acquired by confocal microscopy. *P<0.05; Kruskal-Wallis ANOVA (panels B and D).

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Cell Culture, Confocal Microscopy, Marker, Staining, Fluorescence, Phagocytosis Assay

Journal: PLoS Pathogens

Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

doi: 10.1371/journal.ppat.1007625

Figure Lengend Snippet: Circulating DENV NS1 binds to platelets via TLR4 or other molecules to induce the release of ADP, which in turn elevates P-selectin expression and PS exposure on platelet surfaces leading to platelet activation and enhancement of the platelet aggregation. In addition, NS1-activated platelets are prone to adhere onto endothelium or phagocytosis by macrophages. On the other hand, NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release. All these effects induced by NS1 can contribute to the thrombocytopenia and hemorrhage during DENV infection.

Article Snippet: Recombinant DENV1-4 serotype NS1 proteins used in all experiments were produced by CTK Biotech (San Diego, CA, USA) in Drosophila S2 cells.

Techniques: Expressing, Activation Assay, Infection